Use of phthalide derivatives for the treatment and prevention of diabetes mellitus

ABSTRACT

The present invention relates to the use of a compound as effective agent for the prevention or treatment of diabetes mellitus in a mammal. Said compounds being selected from the group of phthalide derivatives and exhibit excellent blood glucose lowering effects and thus are effective agents in the prevention or treatment of diabetes mellitus in mammals.

The present invention relates to the use of a compound as effectiveagent for the prevention or treatment of diabetes mellitus in a mammal.Said compounds being selected from the group of phthalide derivativesand are useful for the preparation of a pharmaceutical or a dietarycomposition given to a mammal in need thereof for the prevention ortreatment of diabetes mellitus.

Diabetes mellitus defines a complex of metabolic diseases derived frommultiple causative factors and is characterized by impairedcarbohydrate, protein and fat metabolism associated with a deficiency ininsulin secretion and/or insulin resistance. This results in elevatedfasting and postprandial serum glucose that leads to complications ifleft untreated. Four different forms of diabetes mellitus are known, (1)type 1 diabetes mellitus, (2) type 2 diabetes mellitus, (3) theso-called gestational diabetes mellitus, which begins or is recognizedfor the first time during pregnancy, and (4) some other forms which aremainly based on genetic defects.

The two major forms of diabetes mellitus are the type 1 and type 2diabetes mellitus, of which type 2 diabetes mellitus is the mostprevailing form. Type 1 and type 2 diabetes inellitus are associatedwith hyperglycemia, hypercholesterolemia and hyperlipidemia. Theinsensitivity to insulin and absolute insulin deficiency in type 1 and 2diabetes mellitus leads to a decrease in glucose utilization by theliver, muscle and the adipose tissue and to an increase in the bloodglucose levels. Uncontrolled hyperglycemia is associated with increasedand premature mortality due to an increased risk for microvascular andmacrovascular diseases, including nephropathy, neuropathy, retinopathy,hypertension, stroke, and heart disease. Recent evidence showed thattight glycemic control is a major factor in the prevention of thesecomplications in both type 1 and type 2 diabetes mellitus. Therefore,optimal glycemic control by drugs or therapeutic regimens is animportant approach for the treatment of diabetes mellitus.

Type 1 diabetes mellitus is the form of diabetes mellitus which usuallybegins with childhood or puberty and is characterized by an auto-immunedestruction of the insulin-producing β-cells leading to a completedeficiency of insulin secretion. Type 2 diabetes mellitus is the form ofdiabetes mellitus which occurs predominantly in adults in whom adequateproduction of insulin is available for use, yet a defect exists ininsulin-mediated utilization and metabolism of glucose in peripheraltissues. The changes in various tissues associated with type 2 diabetesmellitus exist even before clinical symptoms are detected.

Therapy of type 2 diabetes mellitus initially involves dietary andlifestyle changes, when these measures fail to maintain adequateglycemic control the patients are treated with oral hypoglycemic agentsand/or exogenous insulin. The current oral pharmacological agents forthe treatment of type 2 diabetes mellitus include those that potentiateinsulin secretion (sulphonylurea agents), those that improve the actionof insulin in the liver (biguanide agents), insulin sensitizing agents(thiazolidinediones) and agents which act to inhibit the uptake ofglucose in the gastrointestinal tract (α-glucosidase inhibitors).However, currently available agents generally fail to maintain adequateglycemic control in the long term due to progressive deterioration inhyperglycemia, resulting from progressive loss of pancreatic cellfunction. The proportion of patients able to maintain target glycemiclevels decreases markedly overtime necessitating the administration ofadditionallalternative pharmacological agents. Furthermore, the drugsmay have unwanted side effects and are associated with high primary andsecondary failure rates.

It is known that the peroxisome proliferator-activated receptors (PPARs)play a critical physiological role as lipid sensors and regulators oflipid metabolism. The PPARs are activated to various degrees by highconcentrations of long-chain fatty acids. Synthetic PPAR ligands,including the fibrates and thiazolidinediones, have proven effective inthe treatment of dyslipidemia and diabetes mellitus, especially type 2diabetes mellitus. Mostly, these compounds act with PPARγ, which is oneisoform of the PPAR family, some of them are also able to interact withPPARα and/or PPARδ isoform.

Therefore, although the therapies of choice in the treatment of type 1and type 2 diabetes mellitus are based essentially on the administrationof insulin and of oral hypoglycemic drugs, there is a need for compoundswith minimal side effects for the treatment and prevention of diabetesmellitus. Many patients are interested in alternative therapies whichcould minimize the side effects associated with high-dose of drugs andyield additive clinical benefits. Type 2 diabetes mellitus is aprogressive and chronic disease, which usually is not recognized untilsignificant damage has occurred to the pancreatic cells responsible forproducing insulin and to the cardiovascular system. Therefore, there isalso an increasing interest in the development of a dietary supplementthat may be used to prevent the development of diabetes mellitus inpeople at risk especially in elderly who are at high risk for developingdiabetes inellitus.

We now found that a group of known compounds exhibit excellent bloodglucose lowering effects and thus are effective agents in the preventionor treatment of diabetes mellitus in mammals. These non-toxic compoundsfunction as ligands for PPARγ. Therapeutic effects of these ligandsinclude, but are not limited to, a decrease in the blood glucose level,prevention of obesity, a lowered insulin resistance, delay or preventionof type 2 diabetes mellitus and dyslipidemia, differentiation ofadipocytes, lowered triglyceride levels, and normalized glucosetolerance.

The object of the present invention is the use of a compound representedby formula (I) as antidiabetic agent,

whereinthe dotted line is an optional bond;

-   -   R¹ is butyl or butyryl if R² is hydroxyl but is butyl if R² is        hydrogen; or R¹ and R² taken together are 1-butyildene        optionally substituted by hydroxyl, methyl, or        3-(α,β-dimethylacrylyloxy)-pentylidenyl;    -   X is a residue selected from the group consisting of X1, X2, X3,        X4, and X5;        wherein    -   X is X2, X3or X5 if the dotted line does not signify a bond in        formula (I) above and X is X1, X4 or X5 if the dotted line        signifies a bond in formula (I) above;    -   R³ is hydroxyl or butyryl; and    -   n is 1 or 2.

Thus, in a compound of formula (I) above X is X2, X3, or X5 in case of atetrahydrofuran, i.e., if the dotted line in formula (I) does notsignify a bond whereas X is X1, X4, or X5 in case of a dihydrofuran,i.e., if the dotted line in formula (I) signifies a bond.

The compounds according to formula (I) above as used herein may be alsoin the form of their pharmaceutically acceptable salts.

As used herein, the term “antidiabetic agent” means an agent which iscapable of preventing or treating diabetes mellitus, especially type 2diabetes mellitus, in a mammal which is in need thereof These agents arealso useful for the treatment or prevention of related symptoms.

The term “diabetes mellitus” also includes, but is not limited to,related symptoms such as increased blood glucose level, obesity,increased insulin resistance, hyperlipidemia, dyslipidemia, increase incholesterol (hypercholesterinemia, hypertriglycerinemia),hyperinsulinemia, and impaired glucose tolerance. Impaired glucosetolerance and impaired fasting glucose are the two symptoms referred toas pre-diabetes mellitus. This stage is associated with the so-calledinsulin resistance syndrome also known as syndrome X. Since syndrome Xis directly involved in the pathogenesis of type 2 diabetes mellitus,the compounds used for the present invention are also useful for thetreatment or prevention of syndrome X.

An agonist of PPARγ relates to a small molecule interacting directlywith PPARγ, particularly with its ligand binding domain, and thusactivating the PPARγ.

The compounds as used for the present invention are selected from thegroup of phthalide derivatives, which refer to substituted lactones of2-hydroxymethylbenzoic acid according to IUPAC Rule C-473. This class ofcompounds is based on 1(3H)-isobenzofuranone C₈H₆O₂.

Preferred compounds used as antidiabetic agents are selected from thegroup consisting of (E)-senkyunolide E; senkyunolide C; senkyunolide B;3-butyl-4,5,6,7-tetrahydro-3,6,7-trihydroxy-1(3H)-isobenzofuranone;3-butyl-1(3H)-isobenzofuranone; 3-butylphthalide; 3-butylidenephthalide;chuangxinol; ligustilidiol; senkyunolide F; 3-hydroxy-senkyunolide A;angeloylsenkyunolide F; senkyunolide M; 3-hydroxy-8-oxo-senkyunolide A;ligustilide; 6,7-dihydro-(6S,7R)-dihydroxyligustilide;3a,4-dihydro-3-(3-methylbutylidene)-1(3H)-isobenzofuranone; sedanolide;and cnidilide. The most preferably used compounds are selected from thegroup consisting of ligustilide, 3-butylphthalide,3-butylidenephthalide, and sedanolide. The preferred embodiments arelisted in Table 1. TABLE 1 List of preferred compounds used asantidiabetic agents Structure Name

(E)-Senkyunolide E

Senkyunolide C

Senkyunolide B

3-Butyl-4,5,6,7-tet- rahydro-3,6,7-tri- hydroxy-1(3H)-iso- benzofuranone

3-Butyl-1(3H)-iso- benzofuranone

3-Butylphthalide

3-Butylidenephthalide

Chuangxinol

Ligustilidiol

Senkyunolide F

3-Hydroxy-senkyunolide A

Angeloylsenkyunolide F

Senkyunolide M

3-Hydroxy-8-oxo-senk- yunolide A

Ligustilide

6,7-Dihydro-(6S,7R)-di- hydroxyligustilide

3a,4-Dihydro-3-(3-meth- ylbutylidene)-1(3H)-iso- benzofuranone

Sedanolide

Cnidilide

Thus, it is an object of the present invention to use a compound whichis selected from the group consisting of ligustilide, 3-butylphthalide,3-butylidenephthalide, and sedanolide as antidiabetic agent.

The compounds according to formula (I) as defined above are used for thepreparation of a pharmaceutical or dietary composition for theprevention or treatment of diabetes mellitus. Preferred compounds arerepresented in Table 1. The most preferably used compounds are selectedfrom the group consisting of ligustilide, 3-butylphthalide,3-butylidenephthalide, and sedanolide.

Another aspect of the present invention is the provision of apharmaceutical or dietary composition for use in the treatment orprevention of diabetes mellitus comprising an effective amount of acompound of formula (I) as defined above. Preferred compounds arerepresented in Table 1. The most preferred compounds are selected fromthe group consisting of ligustilide, 3-butylphthalide,3-butylidenephthalide, and sedanolide.

A pharmaceutical composition may further comprise pharmaceuticallyacceptable carriers, excipients or diluents, including, but not limitedto, lubricants, colorants, wetting agents, fillers, disintegrants andflavorants.

A dietary composition may further comprise any known substances whichare normally used and accepted in the preparation of such compositions.

The pharmaceutical or dietary composition may be in the form which isselected from the group consisting of fortified food or feed, beverages,tablets, granules, capsules, pastes, and effervescent formulations. Thepastes may be filled into hard or soft gelatin capsules.

In one aspect, the present invention provides a method for theprevention or treatment of diabetes mellitus and in mammals, said methodcomprising:

-   -   (a) preparing a composition comprising a compound according to        formula (I) as defined above, and    -   (b) administering an effective dose of said composition to a        mammal which is in need thereof.

As used herein, an “effective dose” of the compounds of the presentinvention is an amount which is high enough to effect activation ofPPARγ and thus lowering the blood glucose level in a mammal. A suitabledose is within the range of about 0.01 to about 50 mg/kg bodyweight/day.

The compounds according to formula (I) as defined above and which areused as antidiabetic agents may be isolated by methods known in the art[see, e.g., Beck J. J. and Stermitz F. R., J. Natural Products, Vol. 58,No. 7, pp. 1047-1055, 1995] from various plants such as Angelica glauca,Angelica acutiloba, Angelica sinensis, Angelicae dahuricae, Ligusticumacutilobum, Ligusticum officinale, Ligusticum sinense, Ligusticumwallichii, Cnidium officinale, Rhizoma Chuanxiong, Pleurospermumhookeri, Trachyspermum roxburghianum, Meumn athamanticum, Lomatiumtorreyi, Scutellaria baicalensis, Opopanax chironium, Cenolophiumdenudatum, Coriandrum sativuum, Silaum silaus.The compounds used hereinmay also be of synthetic origin. It is understood that all compounds asused herein are in pure form.

EXAMPLE 1

Effect of Ligustilide on Glucose Uptake of Adipocytes

Unless otherwise stated, the ligustilide as used herein was purchasedfrom GAIA Chemical Corporation, 23 George Washington Plaza,Gaylorsville, Conn. 06755, USA and has a purity of about 95% (purifiedby column chromatography).

C3H10T1/2 cells (ATCC CCL-226) were grown for 5 days to confluence inDMEM supplemented with 10% FBS medium and induced with a mixture ofinsulin, dexamethasone and 3-isobutyl-1-methylxanthine to differentiateinto adipocytes. Nine days after the beginning of induction, cells weretreated for 48-h with ligustilide at different concentrations as shownin Table 2. Glucose uptake was determined using radioactive2-deoxyglucose (10 μM 2-DG in HBS +0.5 μCi/ml of 3[H]-2-DG), measuringglucose uptake in the absence of insulin. Basal glucose uptake wasincreased by 48-h treatment with ligustilide in a dose-dependent manner(Table 2). This increase in glucose uptake was specific, since theincrease was not observed in the presence of phloretin, a knowninhibitor of specific glucose uptake. As a positive control, the knownPPARγ agonist ciglitazone was used in the concentration as indicated inTable 2.

EXAMPLE 2

Effect of 3-Butylphthalide on Glucose Uptake of Adipocytes

Unless otherwise stated, the 3-butylphthalide as used herein waspurchased from Advanced Synthesis Technologies, P.O. Box 437920, SanYsidro, Calif. 92173, USA.

Growing, induction and treatment of C3H10T1/2 cells were exactly asdescribed in Example 1, with the exception that 3-butylphthalide atdifferent concentrations was used instead of ligustilide. An increase ofbasal glucose uptake could be detected as shown in Table 2.

EXAMPLE 3

Effect of 3-Butylidenephthalide on Glucose Uptake of Adipocytes

Unless otherwise stated, the 3-butylidenephthalide as used herein waspurchased from Aldrich Chemical Company, Inc., 1001 West Saint PaulAvenue, Milwaukee, Wis. 53233, USA and has a purity of >96%.

Growing, induction and treatment of C3H10T1/2 cells were exactly asdescribed in Example 1, with the exception that 3-butylidenphthalide atdifferent concentrations was used instead of ligustilide. As shown inTable 2, an increase of the basal glucose uptake could be detected.TABLE 2 Induction of glucose uptake by 48-h treatment with differentcompounds (% of control ± SEM) Concentration Basal glucose Compound [M]uptake Ciglitazone 5 × 10⁻⁵ 381.5 ± 24.4 Ligustilide 5 × 10⁻⁶ 105.9 ±24.4 5 × 10⁻⁵ 131.8 ± 24.4 1 × 10⁻⁴ 175.0 ± 24.4 2 × 10⁻⁴ 294.4 ± 24.43-Butylphthalide 1 × 10⁻⁶  99.3 ± 8.5 1 × 10⁻⁵  97.5 ± 8.5 1 × 10⁻⁴136.7 ± 8.5 3-Butylidenephthalide 1 × 10⁻⁶ 107.0 ± 8.5 1 × 10⁻⁵ 123.8 ±8.5 1 × 10⁻⁴ 137.3 ± 8.5Control: C3H10T1/2 cells treated for 48 h with DMSO at the sameconcentration as compound-treated cells and set at 100%

EXAMPLE 4

Effect of Ligustilide on Differentiation of Adipocytes

C3H10T1/2 cells were grown to confluence as described in Example 1, thentreated for 10 days with insulin alone (negative control) or with amixture of insulin and ligustilide at different concentrations (seeTable 3), with re-feeding with fresh medium and compounds every 48-h.After the 10-day treatment, the cells were stained with oil Red O asfollows: cells were washed 2× in PBS and fixed in 10% formalin at roomtemperature for 1 h. After removal of formalin, 200 μl of oil Red Ostaining solution (3:2 mixture of 0.5% w/v oil Red O stock solution andwater) was applied to each well. The cells were incubated for 20 min atroom temperature, washed twice in 2× PBS and incubated for 10 min with300 μl of isopropanol/well for oil Red O extraction. Quantification ofoil Red O was determined by measuring absorbance at 540 nm (mean OD).Co-treatment of C3H10T1/2 cells with insulin and ligustilide resulted ina higher differentiation of the cells into adipocytes than insulin aloneas represented by a higher amount of oil Red O staining (Table 3). TABLE3 Induction of adipocyte differentiation by 10-day treatment withligustilide Compound Mean OD ± SEM Insulin (2 × 10⁻⁷ M) 0.687 ± 0.34Insulin (2 × 10⁻⁷ M) +  1.71 ± 0.34 ligustilide (5 × 10⁻⁶ M)

EXAMPLE 5

Effect of 3-Butylphthalide on Differentiation of Adipocytes

C3H10T1/2 cells were grown and treated as described in Example 4 withthe exception that 3-butylphthalide was used instead of ligustilide. Themeasurement of adipocyte differentiation using the oil Red O assay wasperformed as described in Example 4. Co-treatment of C3H10T1/2 cellswith insulin and 3-butylphthalide resulted in a higher differentiationof the cells into adipocytes than insulin alone (Table 4). TABLE 4Induction of adipocyte differentiation by 10-day treatment with3-butylphthalide or 3-butylidenephthalide Compound Mean OD ± SEM Insulin(1 × 10⁻⁷ M) 0.14 ± 0.01 Insulin (1 × 10⁻⁷ M) + 0.21 ± 0.013-butylphthalide (1 × 10⁻⁵ M) Insulin (1 × 10⁻⁷ M) + 0.15 ± 0.013-butylidenephthalide (1 × 10⁻⁵ M) Insulin (1 × 10⁻⁷ M) + 0.22 ± 0.013-butylidenephthalide (5 × 10⁻⁵ M) Insulin (1 × 10⁻⁷ M) + 0.28 ± 0.013-butylidenephthalide (1 × 10⁻⁴ M)

EXAMPLE 6

Effect of 3-Butylidenephthalide on Differentiation of Adipocytes

C3H10T1/2 cells were grown and treated as described in Example 4 withthe exception that 3-butylidenephthalide was used instead ofligustilide. The measurement of adipocyte differentiation using the oilRed O assay was performed as described in Example 4. Co-treatment ofC3H10T1/2 cells with insulin and 3-butylidenephthalide resulted in ahigher differentiation of the cells into adipocytes than insulin alone(Table 4).

EXAMPLE 7

Effect of Sedanolide on Differentiation of Adipocytes

Unless otherwise stated, the 3-butylphthalide as used herein waspurchased from Sigma, P.O. Box 14508, St. Louis, Mo. 63178, USA and hasa purity of about >98%.

C3H10T1/2 cells were grown and treated as described in Example 4 withthe exception that sedanolide was used instead of ligustilide. Themeasurement of adipocyte differentiation using the oil Red O assay wasperformed as described in Example 4. Co-treatment of C3H10T1/2 cellswith insulin and sedanolide resulted in a higher differentiation of thecells into adipocytes than insulin alone (Table 5). TABLE 5 Induction ofadipocyte differentiation by 10-day treatment with sedanolide CompoundMean OD ± SEM Insulin (1 × 10⁻⁷ M) 0.16 ± 0.01 Insulin (1 × 10⁻⁷ M) +0.15 ± 0.01 sedanolide (1 × 10⁻⁵ M) Insulin (1 × 10⁻⁷ M) + 0.18 ± 0.01sedanolide (1 × 10⁻⁴ M)

EXAMPLE 7

Effect of Ligustilide on Glucose Tolerance

The efficacy of ligustilide on glucose tolerance was tested in a 7-daystudy in C57BLKS/J db/db mice (n=10/group), a model of late type 2diabetes mellitus with severe hyperglycemia which is widely used todetermine the efficacy of PPARγ ligands.

Male db/db mice were obtained from Jackson Laboratory (Bar Harbor, Me.,USA). Adult mice aged 12 weeks were used in the experiment. Mice werehoused individually in plastic cages with bedding and allowed freeaccess to standard rodent food and tap water. The animal rooms werecontrolled for temperature (24° C.), humidity (55%), and light (12-hlight-dark cycle). The animals were randomized in two groups andligustilide was administered orally to one of the groups for 7 days at adose of 200 mg/kg BW/day. After 7 days of treatment the concentration ofglucose was determined in blood from fed animals, i.e., animals whichwere not restricted from food. After a period of 5 days of treatment anoral glucose tolerance test (OGTT) was performed. For the OGTT mice werefasted overnight and then a 1-g glucose/kg BW solution was orallyadministered. Blood samples were taken before and 15, 30, 45, 60, 90,120, 150, 180 min after the glucose challenge for determination of bloodglucose levels and then the area under the curve (AUC) was determined.Blood glucose was measured by a glucose analyzer (Glucotrend Premium,Roche Diagnostics, Rotkreuz, Switzerland). The blood glucose levels andAUC for the OGTT measurement are given in Table 6. The glucose levels offed animals (see above) were significantly lowered after 7 days ofligustilide treatment.

After 5 days of ligustilide treatment the glucose levels of fastedanimals, i.e., animals with an overnight fasting (see above) weresignificantly decreased as compared to the untreated control group.During the OGTT test the blood glucose levels in the ligustilide treatedanimals were lower at all time points when compared with the controlgroup. Thus, ligustihide significantly reduced the glucose AUC of anOGTT (1 g glucose/kg body weight) on day 5. TABLE 6 Blood Glucose Fasted(mg/dl) Fed (mg/dl) Glucose AUC Control 233 503 69340 Ligustilide (200mg/kg 196 388 51039 BW/day)

1-10. (canceled)
 11. A method of preventing or treating diabetesmellitus in mammals comprising: administering to a mammal an effectivedose of compound represented by formula (I),

wherein the dotted line is an optional bond; R¹ is butyl or butyryl ifR² is hydroxyl but is butyl if R² is hydrogen; or R¹ and R² takentogether are 1-butylidene optionally substituted by hydroxyl, methyl, or3-(α,β-dimethylacrylyloxy)-pentylidenyl; X is a residue selected fromthe group consisting of X1, X2, X3, X4, and X5;

wherein X is X2, X3 or X5 if the dotted line does not signify a bond informula (I) above and X is X1, X4 or X5 if the dotted line signifies abond in formula (I) above; R³ is hydroxyl or butyryl; and n is 1 or 2.12. A method according to claim 11 wherein the compound is selected fromthe group consisting of (E)-senkyunolide E; senkyunolide C; senkyunolideB; 3-butyl-4,5,6,7-tetrahydro-3,6,7-trihydroxy-1(3H)-isobenzofuranone;3-butyl-1(3H)-isobenzofuranone; 3-butylphthalide; 3-butylidenephthalide;chuangxinol; ligustilidiol; senkyunolide F; 3-hydroxy-senkyunolide A;angeloylsenkyunolide F; senkyunolide M; 3-hydroxy-8-oxo-senkyunolide A;ligustilide; 6,7-dihydro-(6S,7R)-dihydroxyligustilide;3a,4-dihydro-3-(3-methylbutylidene)-1(3H)-isobenzofuranone; sedanolide;and cnidilide.
 13. A compound represented by formula (I):

wherein the dotted line is an optional bond; R¹ is butyl or butyryl ifR² is hydroxyl but is butyl if R² is hydrogen; or R¹ and R² takentogether are 1-butylidene optionally substituted by hydroxyl, methyl, or3-(α,β-dimethylacrylyloxy)-pentylidenyl; X is a residue selected fromthe group consisting of X1, X2, X3, X4, and X5;

wherein X is X2, X3 or X5 if the dotted line does not signify a bond informula (I) above and X is X1, X4 or X5 if the dotted line signifies abond in formula (I) above; R³ is hydroxyl or butyryl; and n is 1 or 2.14. A method of making a pharmaceutical or dietary composition for theprevention or treatment of diabetes mellitus comprising: admixing acompound represented by formula (I)

wherein the dotted line is an optional bond; R¹ is butyl or butyryl ifR² is hydroxyl but is butyl if R² is hydrogen; or R¹ and R² takentogether are 1-butylidene optionally substituted by hydroxyl, methyl, or3-(α,β-dimethylacrylyloxy)-pentylidenyl; X is a residue selected fromthe group consisting of X1, X2, X3, X4, and. X5;

wherein X is X2, X3 or X5 if the dotted line does not signify a bond informula (I) above and X is X1, X4 or X5 if the dotted line signifies abond in formula (I) above; R³ is hydroxyl or butyryl; and n is 1 or 2,with a pharmaceutically acceptable carrier, an excipient, a diluent, afortified food or feed, or a beverage.
 15. A pharmaceutical or dietarycomposition for the treatment or prevention of diabetes mellituscomprising an effective amount of a compound of formula (I),

wherein the dotted line is an optional bond; R¹ is butyl or butyryl ifR² is hydroxyl but is butyl if R2 is hydrogen; or R¹ and R² takentogether are 1-butylidene optionally substituted by hydroxyl, methyl, or3-(α,β-dimethylacrylyloxy)-pentylidenyl; X is a residue selected fromthe group consisting of X1, X2, X3, X4, and X5;

wherein X is X2, X3 or X5 if the dotted line does not signify a bond informula (I) above and X is X1, X4 or X5 if the dotted line signifies abond in formula (I) above; R³ is hydroxyl or butyryl; and n is 1 or 2.16. A composition according to claim 15 comprising a compound selectedfrom the group consisting of (E)-senkyunolide E; senkyunolide C;senkyunolide B;3-butyl-4,5,6,7-tetrahydro-3,6,7-trihydroxy-1(3H)-isobenzofuranone;3-butyl-1 (3H)-isobenzofuranone; 3-butylphthalide;3-butylidenephthalide; chuangxinol; ligustilidiol; senkyunolide F;3-hydroxy-senkyunolide A; angeloylsenkyunolide F; senkyunolide M;3-hydroxy-8-oxo-senkyunolide A; ligustilide;6,7-dihydro-(6S,7R)-dihydroxyligustilide;3a,4-dihydro-3-(3-methylbutylidene)-1(3H)-isobenzofuranone; sedanolide;and cnidilide.
 17. A composition according to claim 15 comprising acompound selected from the group consisting of ligustilide,3-butylphthalide, 3-butylidenephthalide, and sedanolide.
 18. Acomposition according to claim 15 further comprising a pharmaceuticallyacceptable carrier, excipient, or diluent.
 19. A composition accordingto claim 16 further comprising a pharmaceutically acceptable carrier,excipient, or diluent.
 20. A composition according to claim 17 furthercomprising a pharmaceutically acceptable carrier, excipient, or diluent.21. A composition according to claim 15 in a form selected from thegroup consisting of a fortified food or feed, a beverage, a tablet, agranule, a capsule, a paste, and an effervescent formulation.
 22. Acomposition according to claim 16 in a form selected from the groupconsisting of a fortified food or feed, a beverage, a tablet, a granule,a capsule, a paste, and an effervescent formulation.
 23. A compositionaccording to claim 17 in a form selected from the group consisting of afortified food or feed, a beverage, a tablet, a granule, a capsule, apaste, and an effervescent formulation.
 24. A composition according toclaim 18 in a form selected from the group consisting of a fortifiedfood or feed, a beverage, a tablet, a granule, a capsule, a paste, andan effervescent formulation.
 25. A method for the prevention ortreatment of diabetes mellitus in mammals, said method comprising: (a)preparing a composition comprising an effective amount of a compound offormula (I),

wherein the dotted line is an optional bond; R¹ is butyl or butyryl ifR² is hydroxyl but is butyl if R2 is hydrogen; or R¹ and R² takentogether are 1-butylidene optionally substituted by hydroxyl, methyl, or3-(α,β-dimethylacrylyloxy)-pentylidenyl; X is a residue selected fromthe group consisting of X1, X2, X3, X4, and X5;

wherein X is X2, X3 or X5 if the dotted line does not signify a bond informula (I) above and X is X1, X4 or X5 if the dotted line signifies abond in formula (I) above; R³ is hydroxyl or butyryl; and n is 1 or 2,and (b) administering an effective dose of said composition to a mammalwhich is in need thereof.
 26. A method according to claim 25 wherein thecompound of formula 1 is selected from the group consisting of(E)-senkyunolide E; senkyunolide C; senkyunolide B;3-butyl-4,5,6,7-tetrahydro-3,6,7-trihydroxy-1(3H)-isobenzofuranone;3-butyl-1(3H)-isobenzofuranone; 3-butylphthalide; 3-butylidenephthalide;chuangxinol; ligustilidiol; senkyunolide F; 3-hydroxy-senkyunolide A;angeloylsenkyunolide F; senkyunolide M; 3-hydroxy-8-oxo-senkyunolide A;ligustilide; 6,7-dihydro-(6S,7R)-dihydroxyligustilide;3a,4-dihydro-3-(3-methylbutylidene)-1(3H)-isobenzofuranone; sedanolide;and cnidilide.
 27. A method according to claim 25 wherein the compoundof formula 1 is selected from the group consisting of ligustilide,3-butylphthalide, 3-butylidenephthalide, and sedanolide.
 28. A methodaccording to claim 25 wherein the composition further comprises apharmaceutically acceptable carrier, excipient, or diluent.
 29. A methodaccording to claim 25 wherein the composition is in a form selected fromthe group consisting of a fortified food or feed, a beverage, a tablet,a granule, a capsule, a paste, and an effervescent formulation.